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Differential association of membrane-bound and non-membrane-bound polysomes with the cytoskeleton
G Zambetti1, L Wilming, E G Fey
1Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
Abstract:
We report here a differential release of specific mRNAs from the cytoskeleton by cytochalasin D treatment. Non-membrane-bound polysomal mRNAs, such as histone mRNA and c-fos mRNA, are readily released from the cytoskeleton of HeLa cells during cytochalasin D treatment. Over 90% of H3 and H4 histone mRNA is associated with the cytoskeleton in control cells and only 25% in cells treated with cytochalasin D (40 micrograms/ml). In contrast, the membrane-bound polysomal mRNAs for HLA-B7 and chorionic gonadotropin-alpha are inefficiently released from the cytoskeletal framework by cytochalasin D alone; approximately 98% of the HLA-B7 mRNA in control cells is associated with the cytoskeleton, whereas approximately 65% of the HLA-B7 mRNA is retained on the cytoskeleton in cells treated with cytochalasin D (40 micrograms/ml). Disruption of polysome structure with puromycin during cytochalasin D treatment results in the efficient release of HLA-B7 mRNA from the cytoskeleton. Under these conditions, only 25% of the HLA-B7 mRNA remains associated with the cytoskeletal framework. Thus, membrane-bound polysomes appear to be attached to the cytoskeleton through a cytochalasin D-sensitive site as well as through association with the nascent polypeptide and/or ribosome. These results demonstrate a complex association of polysomes with the cytoskeleton and elements of the endoplasmic reticulum.
Insights
Cytochalasin D treatment differentially releases specific mRNAs from the cytoskeleton. Non-membrane-bound mRNAs like histone mRNA are easily released, while membrane-bound mRNAs show complex cytoskeletal association.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The cytoskeleton plays a crucial role in cellular organization and mRNA localization.
- Understanding mRNA-cytoskeleton interactions is key to deciphering gene expression regulation.
Purpose of the Study:
- To investigate the differential release of specific mRNAs from the cytoskeleton upon cytochalasin D treatment.
- To elucidate the mechanisms underlying the association of membrane-bound polysomes with the cytoskeleton.
Main Methods:
- HeLa cells were treated with cytochalasin D (40 µg/ml) to disrupt the cytoskeleton.
- Puromycin was used in conjunction with cytochalasin D to disrupt polysome structure.
- The association of specific mRNAs (histone, c-fos, HLA-B7, chorionic gonadotropin-alpha) with the cytoskeleton was quantified before and after treatment.
Main Results:
- Cytochalasin D treatment readily released non-membrane-bound polysomal mRNAs (histone, c-fos) from the cytoskeleton.
- Membrane-bound polysomal mRNAs (HLA-B7, chorionic gonadotropin-alpha) were inefficiently released by cytochalasin D alone.
- Disruption of polysomes with puromycin during cytochalasin D treatment led to efficient release of HLA-B7 mRNA, indicating a dual attachment mechanism.
Conclusions:
- Polysome association with the cytoskeleton is complex, involving both cytochalasin D-sensitive sites and interactions with nascent polypeptides/ribosomes.
- Membrane-bound polysomes exhibit a distinct cytoskeletal attachment mechanism compared to non-membrane-bound polysomes.
- These findings highlight the intricate interplay between the cytoskeleton, endoplasmic reticulum, and mRNA localization in cellular processes.